Zeiss LSM 510, Manual

Page: 565 / 606

MULTIPHOTON LASER SCANNING MICROSCOPY
LSM 510 META NLO Contents Carl Zeiss
03/06
B 45-0021 e 9-3
CONTENTS
Page
9 MULTIPHOTON LASER SCANNING MICROSCOPY..........................................................9-5
9.1 Preface ................................................................................................................. 9-5
9.2 Introduction to Multiphoton Laser Scanning Microscopy........................................ 9-6
9.2.1 Multiphoton excitation – How does it work? ......................................................................9-6
9.2.2 Increased signal-to-noise, enhanced vitality, and deep optical sectioning in MPLSM............9-8
9.2.3 Drawbacks of using NIR light for microscopy ......................................................................9-9
9.2.4 Achieving Efficient Multiphoton Excitation using Ultrafast lasers .......................................9-10
9.2.5 Optimizing the Peak Intensity without Frying the Samples ................................................9-11
9.3 Using the LSM 510 NLO direct coupled system: Practical considerations for ................
optimal imaging.................................................................................................. 9-13
9.3.1 Coupling an ultrafast laser to the LSM 510 NLO ...............................................................9-13
9.3.2 Alignment of the ultrafast laser into the LSM 510 NLO scan head ....................................9-14
9.3.2.1 Quick alignment protocol.................................................................................................9-14
9.3.2.2 Advanced alignment protocol ..........................................................................................9-16
9.3.2.3 Tips on maintaining alignment with the scan head ...........................................................9-17
9.3.3 Objectives recommended for Multiphoton Excitation........................................................9-18
9.3.4 Choosing Fluorescent Probes for MPLSM..........................................................................9-20
9.3.5 Samples your mother should have warned you about.......................................................9-22
9.4 Troubleshooting Checklist ................................................................................... 9-25
9.4.1 No image being produced................................................................................................9-25
9.4.2 Poor image quality ...........................................................................................................9-26
9.5 References .......................................................................................................... 9-27